This project involves an investigation of the molecular mechanism of enterotoxin B (SEB) production and its control in Staphylococcus aureus. This includes the determination of the genetic location of the toxin determinants and isolation of the toxin gene(s) by molecular cloning. Two approaches will be used for this purpose. The first will consist of shotgun cloning of restriction digests of chromosomal DNA onto a suitable vector plasmid carrying antibiotic resistance markers. Transformants for resistance to the appropriate antibiotic will be plated on agar containing anti-SEB antiserum and screened for halos indicative of the SEB-antibody-antigen reaction. The second cloning stragegy will involve the transposon Tn551 insertions that inactivate SEB production. After random Tn551 (carrying erythromycin resistance) insertions into the chromosome, shotgun cloning will be carried out and the clones will be selected for Emr. Clones obtained will be UV induced for precise excision of Tn551, and the Ems clones would be checked for SEB production. Once the toxin gene(s) has been cloned onto a vector plasmid its DNA sequence will be determined and an in vitro translation system will be established for the synthesis of SEB and this system will be used to identify the elements that control the toxin synthesis. We have found that the toxin gene(s) are chromosomal and very labile, probably similar to some mobile genetic elements in Staphylococcus aureus that we refer to as hitch-hiking transposons.